Optical assay device with pneumatic sample actuation

ABSTRACT

This invention relates generally to devices and methods for performing optical and electrochemical assays and, more particularly, to test devices, e.g., cartridges, methods and systems, wherein the test devices have an entry port configured to receive a test sample into a holding chamber; a first conduit having at least one lateral flow test strip; and a displacement device, such as a pneumatic pump, configured to move a portion of said test sample from said holding chamber into said first conduit. The present invention is particularly useful for performing immunoassays and/or electrochemical assays at the point-of-care.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/163,896, filed May 25, 2016, which is a divisional of Ser.No. 13/724,363, filed Dec. 21, 2012, now U.S. Pat. No. 9,377,475 issuedon Jun. 28, 2016, which claims priority to U.S. Provisional ApplicationNo. 61/579,822 filed on Dec. 23, 2011, the entireties of which arehereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to devices and methods for performingoptical and electrochemical assays and, more particularly, to testdevices, systems and methods wherein the test devices include adisplacement device configured to move a portion of a test sample from aholding chamber to a conduit containing at least one lateral flow teststrip.

BACKGROUND OF THE INVENTION

A multitude of laboratory tests for analytes of interest are performedon biological samples for diagnosis, screening, disease staging,forensic analysis, pregnancy testing, drug testing, and other reasons.While a few qualitative tests have been reduced to simple kits for thepatient's home use, the majority of quantitative tests still require theexpertise of trained technicians in a laboratory setting usingsophisticated instruments. Laboratory testing increases the cost ofanalysis and delays the results. In many circumstances, delay can bedetrimental to a patient's condition or prognosis. In these criticalsituations and others, it would be advantageous to be able to performsuch analyses at the point of care, accurately, inexpensively, and witha minimum of delay.

Devices capable of performing such analyses include a disposable sensingdevice for measuring analytes in a sample of blood, which is disclosedby Lauks et al. in U.S. Pat. No. 5,096,669. Other related devices aredisclosed by Davis et al. in U.S. Pat. Nos. 5,628,961 and 5,447,440 fora clotting time. The disclosed devices comprise a reading apparatus anda cartridge that fits into the reading apparatus for measuring analyteconcentrations and viscosity changes in a sample of blood as a functionof time. However, a potential problem with such disposable devices isvariability of fluid test parameters from cartridge to cartridge due tomanufacturing tolerances or machine wear. Methods to overcome thispotential problem using automatic flow compensation controlled by areading apparatus using conductimetric sensors located within thecartridge are disclosed by Zelin in U.S. Pat. No. 5,821,399. U.S. Pat.Nos. 5,096,669, 5,628,961, 5,447,440, and 5,821,399 are herebyincorporated in their respective entireties by reference.

Antibodies are extensively used in the analysis of biological analytes.A variety of different analytical approaches have been employed todetect, either directly or indirectly, the binding of an antibody to itsanalyte. Various alternative assay formats (other than those used intypical research laboratories, such as Western blotting) have beenadopted for quantitative immunoassays, which are distinguished fromqualitative immunoassay kits, such as pregnancy testing kits. As anexample of antibody use, Swanson et al., U.S. Pat. No. 5,073,484,disclose a method in which a fluid-permeable solid medium has reactionzones through which a sample flows. A reactant that is capable ofreacting with the analyte is bound to the solid medium in a zone.

However, most of the methods currently available for quantitativeimmunoassays either are operated manually or require bulky machinerywith complex fluidics because the quantitative immunoassays typicallyrequire multiple steps (e.g., a binding step followed by a rinse stepwith a solution that may or may not contain a second reagent). Anexample of the latter approach is provided in Holmstrom, U.S. Pat. No.5,201,851, which discloses methods providing complex fluidics for verysmall volumes on a planar surface. Additionally, photomultipliers,phototransistors and photodiodes have been discussed in the context ofimmunoassay development. See, e.g., jointly owned Davis et al., U.S.Pat. No. 8,017,382, the entirety of which is incorporated herein byreference.

Microfabrication techniques (e.g. photolithography and plasmadeposition) are known for construction of multilayered sensor structuresin confined spaces, e.g., the confined spaces of cartridges for theabove-disclosed devices. Methods for microfabrication of electrochemicalimmunosensors, for example on silicon substrates, are disclosed byCozzette et al. in U.S. Pat. No. 5,200,051, the entirety of which isincorporated herein by reference. These include dispensing methods,methods for attaching biological reagent, e.g., antibodies, to surfacesincluding photoformed layers and microparticle latexes, and methods forperforming electrochemical assays.

Additionally, jointly owned Davis et al., U.S. Pat. No. 7,419,821, theentirety of which is incorporated herein by reference, discloses asingle-use cartridge designed to be adaptable to a variety of real-timeassay protocols, preferably assays for the determination of analytes inbiological samples using immunosensors or other ligand/ligandreceptor-based biosensor embodiments. The cartridge provides featuresfor processing a metered portion of a sample, for precise and flexiblecontrol of the movement of a sample or second fluid within thecartridge, for the amending of solutions with additional compoundsduring an assay, and for the construction of immunosensors capable ofadaptation to diverse analyte measurements.

Furthermore, Davis et al., U.S. Pat. No. 7,419,821, discloses mobilemicroparticles capable of interacting with an analyte and ways oflocalizing the microparticles onto a sensor, e.g., with a magnetic fieldor a porous filter element. However, to date, one step immunoassays withlimited or no wash steps have not been used for antigens where thepresence of endogenous related antigens create high backgrounds thatconfound detection results. This is particularly true when theendogenous antigens are found at high molar concentrations in excess ofthe antigen of interest, which is common for some disease conditions.

Immunoassays for the determination of analytes in biological samples, asdiscussed above, may include a variety of assay types such as lateralflow tests. Typical lateral flow tests are a type of immunoassay inwhich the test sample flows along a solid substrate via capillaryaction. For example, once the test sample is applied to the substrate,the sample may traverse the substrate via capillary action encounteringa colored reagent, which mixes with the sample, and subsequently to testlines or zones that have been pretreated with an antibody or antigen.The colored reagent can become bound at the test lines or zonesdepending upon the presence or absence of the analyte in the testsample. General background for lateral flow technology may be found inthe following: (i) Brown et al., U.S. Pat. No. 5,160,701, disclose asolid-phase analytical device and method; (ii) Cole et al., U.S. Pat.No. 5,141,850, disclose a porous strip for an assay device; (iii) Fan etal., WO 91/012336, disclose an immunochromatographic assay and method;(iv) Fitzpatrick et al., U.S. Pat. No. 5,451,504, disclose a method anddevice for detecting the presence of analyte in a sample; (v) Imrich etal., U.S. Pat. No. 5,415,994, disclose a lateral flow medical diagnosticassay device; (vi) Kang et al., U.S. Pat. No. 5,559,041, discloseimmunoassay devices and materials; (vii) Koike, EP 0505636, disclosesimmunochromatographic assay methods; (viii) May et al., WO 88/008534,disclose various immunoassay devices; (ix) Rosenstein, EP 0284232,discloses details of solid phase assays; (x) Sommer, U.S. Pat. No.5,569,608, discloses quantitative detection of analytes onimmunochromatographic strips; and (xi) Allen et al., U.S. Pat. No.5,837,546, disclose electronic assay devices and methods.

Lateral flow test devices have also been combined with barcode systemsfor the determination of information pertinent to the lateral flow test,e.g., the identification of the analyte being tested and the patient.General background for the use of barcodes on lateral flow and othertypes of devices for testing clinical samples may be found in thefollowing: (i) Markart et al., U.S. Pat. No. 4,509,859; (ii) Poppe etal., U.S. Pat. No. 4,592,893; (iii) Ruppender, U.S. Pat. No. 4,510,383;(iv) Crosby, U.S. Pat. No. 6,770,487; (v) commercial items, e.g.,Ektachem™ and Reflotron™ products; (vi) Piasio et al., WO 2010017299;(vii) Broich et al., U.S. Pat. No. 7,267,799; (viii) Bhullar et al.,U.S. Pat. No. 6,814,844 and McAleer et al. U.S. Pat. No. 5,989,917; (ix)Rehm, EP 1225442; (x) Eyster et al., EP 1359419, and (xi) Howard, III etal., U.S. Pat. No. 5,408,535; (xii) Babu et al., U.S. Patent ApplicationPublication No. 2007/0202542; and (xiii) Nazareth et al., U.S. Pat. No.7,763,454, and (ixx) Nazareth et al., U.S. Patent ApplicationPublication No. 2010/0240149.

Lateral flow assays also have been adapted to include time-resolvedluminescence detection. Time-resolved luminescence detection techniquesmay have higher detection sensitivity than conventional luminescencetechniques (e.g., fluorescence and phosphorescence) due to highersignal-to-noise ratios. Compared with standard luminescence detectionmethods that separate the luminescence of interest from the backgroundsignal through wavelength differences, time-resolved luminescencetechniques separate the luminescence of interest from the backgroundsignal through lifetime differences. Time-resolved luminescencetechniques operate by exciting a luminescent label of a longluminescence lifetime with a short pulse of light, and waiting a briefperiod of time (e.g., 10 μs) for the background and other unwanted lightto decay to a low level before collecting the remaining long-livedluminescence signal. General background for lateral flow assays capableof time-resolved luminescence detection may be found in the following:Song and M. Knotts, “Time-Resolved Luminescent Lateral Flow AssayTechnology,” Analytica Chimica Acta, vol. 626, no. 2, pp. 186-192,(2008), and Song et al. U.S. Patent Application Publication No.2009/0314946.

As an alternative to the lateral flow test formats, immunoassays mayalso include microarray techniques, which rely on optical detection.Microarrays are an array of very small samples of purified DNA orprotein target material arranged typically as a grid of hundreds orthousands of small spots on a substrate. When the microarray is exposedto selected probe material, the probe material selectively binds to thetarget spots only where complementary bonding sites occur. Subsequentscanning of the microarray by a scanning instrument may be used toproduce a pixel map of fluorescent intensities, which can be analyzedfor quantification of fluorescent probes and hence the concentration ofan analyte. General background for microarray techniques may be found,for example, in Schermer et al., U.S. Pat. No. 6,642,054, whichdiscloses microarray spotting instrumentation that incorporates sensorsfor improving the performance of microarrays.

Therefore, there exists within the field of analyte sensing, and inparticular for applications in which analytes must be determined withinbiological samples such as blood, a need for devices that can rapidlyand simply determine the presence and/or concentration of analytes atpatient point-of-care, and can be performed by less highly trained staffthan is possible for conventional laboratory-based testing. It would,for example, be of benefit in the diagnosis and treatment of criticalmedical conditions for the attending physician or nurse to be able toobtain clinical test results without delay. The need also exists forimproved devices that are adaptable to the determination of a range ofanalytes.

SUMMARY OF THE INVENTION

The invention is directed to testing devices, e.g., test cartridges,systems, methods and computer program products for optical and/orelectrochemical analyte testing. In one embodiment, the device comprisesan entry port configured to receive a test sample into a holdingchamber; a first conduit comprising at least one lateral flow teststrip; and a displacement device, preferably a pneumatic pump,configured to move a portion of said test sample from said holdingchamber into said first conduit.

In another embodiment, the invention is to a testing system comprising atesting device comprising: (i) an entry port configured to receive atest sample into a holding chamber; a first conduit comprising at leastone lateral flow test strip; and (ii) a displacement device, preferablya pneumatic pump, configured to move a portion of said test sample fromsaid holding chamber into said first conduit. In this embodiment, thetesting system further comprises an instrument comprising a receivingport configured to receive and engage said testing device. Theinstrument preferably further comprises an optical imager for reading anoptical signal from said at least one lateral flow test strip. In oneaspect, the optical imager is configured to simultaneously read saidoptical signal and a bar code on said testing device.

The device optionally further comprises a position sensor configured toprovide a position of said portion of said test sample based onconductance of said test sample in at least one predetermined locationof said testing device. For example, in one aspect, the reader device orinstrument further comprises a position detector configured to determinea position of said portion of said test sample in said testing device.The position detector is optionally an electrical circuit that isconfigured to determine the position of said portion of said test samplebased on conductance of said test sample in at least one predeterminedlocation of said testing device. In another aspect, the positiondetector is an imager that is configured to determine the position ofsaid portion of said test sample based on a captured image of said testsample passing through at least one predetermined location of saidtesting device. In one aspect, the device preferably further comprisesat least one imaging area configured to provide a position of saidportion of said test sample based on a captured image of said testsample passing through said at least one imaging area.

The device may also further comprise a labeling zone and a wastechamber. In this aspect, the displacement device preferably is furtherconfigured to move said portion of said test sample from said holdingchamber to said labeling zone. The displacement device may be furtherconfigured to move said portion of said test sample from said labelingzone to said first conduit, and may be further configured to move saidportion of said test sample from said first conduit to said wastechamber.

In some embodiments, the device further comprises a second conduitcomprising at least one lateral flow test strip. In this aspect, thedisplacement device may be further configured to move said portion ofsaid test sample from said holding chamber into said first conduit andsaid second conduit. In another aspect, the device further comprises asecond conduit comprising at least one electrochemical sensor. In thisaspect, the displacement device may be further configured to move saidportion of said test sample from said holding chamber into said firstconduit and said second conduit.

The reader instrument optionally further comprises a processorconfigured to control a displacement device, e.g., a pneumatic pump, toactuate said displacement device based on the determined position ofsaid portion of said test sample. The processor, for example, may befurther configured to control the displacement device, e.g., pneumaticpump, to move said portion of said test sample, optionally from theholding chamber to a labeling zone. The processor optionally is furtherconfigured to control the displacement device to move said portion ofsaid test sample from said labeling zone to said first conduit. Theprocessor optionally is further configured to control the displacementdevice to move said portion of said test sample from said first conduitto said waste chamber. In one aspect, the processor is furtherconfigured to control the displacement device to move said portion ofsaid test sample from said holding chamber into said first conduit andsaid second conduit.

In another embodiment, the invention is to a computer system for testingan analyte, the system comprising a CPU, a computer readable memory anda computer readable storage media; first program instructions to actuatea pneumatic pump using a processor to move a portion of a test sample ina testing device from a holding chamber into a first conduit, said firstconduit comprising a detection zone including a plurality of captureantibodies; and second program instructions to capture an image of saiddetection zone, wherein said first and said second program instructionsare stored on the computer readable storage media for execution by theCPU via the computer readable memory.

The computer system may further comprise third program instructions toactuate said pneumatic pump to move another portion of said test samplein said testing device into a second conduit; and fourth programinstructions to determine an electrochemical characteristic of saidanother portion of said test sample in said second conduit. In oneaspect, the computer system further comprises third program instructionsto detect a location of said portion of said test sample in said testingdevice at a predetermined location; and fourth program instructions toactuate said pneumatic pump to move said portion of said test sampleupon detection of said portion of said test sample at the predeterminedlocation.

In another embodiment, the invention is to an analyte test cartridgecomprising: a sample entry port configured to receive a test sample; asample holding chamber; a labeled antibody for an analyte in said testsample; a displacement device configured to move said test samplethrough at least a portion of said test cartridge; a conduit; and aporous plug with a capture antibody for said analyte. The sample holdingchamber is connected to said closable sample entry port, saiddisplacement device, and said conduit. The labeled antibody ispreferably coated on at least a portion of said conduit, and the labeledantibody is preferably configured to dissolve into said test sample. Theporous plug with said capture antibody is preferably positioned in saidconduit. The label is preferably an optically detectable moiety. Thecartridge may further comprise a waste chamber positioned subsequent tosaid porous plug. The porous plug is preferably aligned with an opticaldetector in an instrument, e.g., a reader instrument. In one embodiment,the cartridge further comprises a plasma separator positioned in atleast a portion of said sample holding chamber proximate to said sampleentry port. In this aspect, the test sample may be whole blood whenreceived at said sample entry port and said test sample may be plasmasubsequent to said sample holding chamber.

In another embodiment, the invention is to a cartridge for detection ofan analyte in a sample, the cartridge comprising: an entry port forreceiving said sample into a holding chamber; a conduit with a beadreagent that is configured to dissolve into said sample; a wickingmatrix comprising an immobilized antibody that is configured to bindwith said analyte; and a displacement device configured to move saidsample into said conduit, mix said bead reagent with said sample to forma complex of said analyte with said bead reagent, and move said sampleto said wicking matrix to capture said complex.

In another embodiment, the invention is to a method of performing ananalyte assay comprising: introducing a sample into a sample holdingchamber of a cartridge through a sample entry port; activating a pump tomove at least a portion of said sample into a conduit; oscillating saidsample to dissolve an optically labeled antibody into said sample and topermit binding of said optically labeled antibody with an analyte toform a complex; waiting a predetermined amount of time; activating saidpump after waiting said predetermined amount of time to move said sampleto a porous plug in said conduit, wherein at least a portion of saidporous plug comprises an immobilized antibody configured to bind to saidcomplex; and activating an optical detector aligned with said porousplug to determine a presence or amount of retained optically labeledantibody.

In another embodiment, the invention is to a computer program productcomprising a computer usable storage medium having readable program codeembodied in the storage medium, wherein the computer program productincludes at least one component operable to: actuate a pneumatic pump tomove a portion of a test sample in a testing device from a holdingchamber into a conduit, a portion of said conduit comprising a captureantibody; and actuate an imager aligned with said portion of saidconduit to capture an image of said portion of said conduit.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood in view of the followingnon-limiting figures.

FIG. 1A shows a top perspective view of an electrochemical cartridge inan open position prior to being folded;

FIG. 1B shows a top perspective view of an optical cartridge in an openposition prior to being folded;

FIG. 2 is an illustrative external environment for implementing theinvention in accordance with some embodiments of the invention;

FIGS. 3A-3D show an instrument port for accepting and reading opticaland electrochemical test devices;

FIG. 4 shows a set of six test strips at a range of concentrations asboth a standard electronic image and using an imager chip;

FIG. 5 shows the imager chip traces in a single plot of intensity versuspixel position;

FIG. 6 shows an orientation of optical elements in accordance with someembodiments of the invention;

FIG. 7 shows a conceptual representation of a lateral flow test strip;

FIGS. 8A-8C show images of lateral flow assay strips at various stagesof testing;

FIG. 9 shows processing of pixel values from image data into graphicalplots;

FIGS. 10A-10C show graphical plots of single intensity at various stagesof testing;

FIGS. 11A-11C show processing of images and signals resulting in adifference image and signal in accordance with some embodiments of theinvention;

FIGS. 12A-12C show processing of images and signals resulting in adifference image and signal in accordance with alternative or additionalembodiments of the invention;

FIG. 13 shows a plot of ratios of measured peaks in accordance withpreferred embodiments of the invention;

FIG. 14 shows a lateral flow test device adapted to mate with aninstrument port;

FIGS. 15A-15C show side, plan and front views, respectively, of alateral flow test device adapted to mate with an instrument port;

FIGS. 16A-16C show top and bottom perspective views and a side view,respectively, of an optical cartridge in accordance with someembodiments of the invention;

FIGS. 17A and 17B show microarray test devices adapted to mate with aninstrument port;

FIG. 18 shows a fluorescent image of a test device with a microspotarray;

FIG. 19 shows quantum dot features that may be used alternatively in thearray of FIG. 18;

FIG. 20 shows a lateral flow strip cartridge with impedance detectionand fluid position tracking;

FIG. 21 shows a microspot array cartridge with impedance detection andfluid position tracking;

FIG. 22 shows a test device with pneumatic mixing of a labeled antibodywith a sample further comprising a lateral flow capture zone;

FIG. 23 shows a test device with pneumatic mixing of a labeled antibodywith a sample further comprising multiple lateral flow capture zones;

FIG. 24 shows another embodiment of a test device with pneumatic mixingof a labeled antibody with a sample further comprising lateral flowcapture zones in multiple channels;

FIG. 25 shows another embodiment of a test device with pneumatic mixingof a labeled antibody with a sample further comprising lateral flowcapture zones in one channel and electrochemical detectors in anotherchannel;

FIG. 26 shows a test device comprising a test sample separator inaccordance with aspects of the invention;

FIGS. 27A-27D show developed images for various concentrations ofanalyte;

FIGS. 28A-28D show processed images of experiments conducted at variousconcentrations; and

FIGS. 29A-29D show processed signals for experiments conducted atvarious concentrations.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The present invention relates to reader devices that are operable withoptical and/or electrochemical assay systems and to novel cartridges foruse with such reader devices. More particularly, the present inventionrelates to immunoassays, and devices and methods for performingimmunoassays and/or electrochemical assays, preferably in thepoint-of-care setting. The present invention advantageously providesaccurate optical and/or electrochemical test results using a singlepoint-of-care reader device.

In one embodiment, the invention is to a reader device having acartridge receiving port configured to accept multiple cartridge types,such as an optical cartridge and/or an electrochemical assay cartridge.In another embodiment, the invention is to a cartridge comprisingoptical and electrochemical assay systems. In further embodiments, theinvention is to a cartridge for optical detection of the results of alateral flow test, e.g., in a qualitative (e.g., providing a positive ornegative test result), semi-quantitative manner (e.g., wherein thedarkness of an optical signal correlates to approximate analyteconcentration), or quantitative manner. In another embodiment, theinvention is to a cartridge for optical detection of the results of amicroarray. In accordance with some aspects of invention, the cartridgemay be further provided with an integrated means for sample actuation.The cartridge may also be provided with an integrated test sampleseparator.

FIG. 1A shows an exemplary cartridge 100, e.g., an i-STAT™ cartridgesold by Abbott Point of Care Inc., Princeton, N.J., USA, that may beemployed with the reader devices of the present invention. Jointly ownedDoyle et al., U.S. Patent Application Publication No. 2011/0150705, theentirety of which is incorporated herein by reference, provides adiscussion of the structural features of the cartridge 100.Specifically, the cartridge 100 comprises an electrochemical assaysystem 110 including electrochemical sensors. In some embodiments of thepresent invention, the cartridge 100 may be modified either to replacethe electrochemical assay system 110 with an optical assay system or toinclude the optical assay system along with the electrochemical assaysystem 110.

For example, FIG. 1B provides an exemplary cartridge 120 comprising anoptical assay system in accordance with some embodiments of theinvention. Specifically, the cartridge 120 may include a cover 122 witha sample input port 125, a base 126 that supports a lateral flow teststrip 128, and an optional transparent tape or film 130 that covers anoptical test window in the base 126 for optical detection of an analytedetected by the lateral flow test strip 128. In some embodiments, notshown, the cartridge 120 may also include an electrochemical assaysystem in addition to the lateral flow test strip 130, as discussed indetail below. In accordance with some aspects of the invention, thecartridges comprise registration features for mating with a port in thereader device to hold and align the cartridge with certain readerfeatures. The reader features aligned with the cartridge may comprise anelectrical connector for reading an electrochemical signal of acartridge of the type shown in FIG. 1A, and/or an imager for reading anoptical signal from a detection zone of a cartridge of the type shown inFIG. 1B, as discussed in detail below.

In further embodiments, the cartridge 120 may also be modified toinclude a barcode for determining information pertaining to thecartridge, e.g., the identification of an analyte being tested and/orthe patient. In accordance with further aspects of the above-mentionedembodiments, the cartridge may also be modified to include a pneumaticpump and/or a test sample separator (not shown), as discussed in detailbelow. Although some aspects of the invention are disclosed with respectto the cartridges shown in FIGS. 1A and 1B, one of ordinary skill in theart would understand that the concepts discussed herein have manyapplications and could be implemented in a wide variety of systems anddevices, e.g., the i-STAT cartridge as disclosed in Lauks et al., U.S.Pat. No. 5,096,669, the entirety of which is incorporated herein byreference.

Reader Device

In some embodiments, the invention is to a reader device, e.g., acomputing device 215 (as discussed with respect to FIG. 2), that isprovided for receiving one or more different types of biological sampletesting cartridges (as discussed with respect to FIGS. 1A and 1B)through a cartridge receiving port.

FIG. 3A shows an exemplary reader 300 comprising a receiving port 310 ina housing 320. In accordance with some aspects of the invention, thereceiving port 310 may be configured to accept multiple cartridge types,including, for example, one or more of: (i) optical test devicescomprising qualitative or semi-quantitative lateral flow test systems,(ii) optical test devices comprising qualitative or semi-quantitativemicrospot array test systems, (iii) electrochemical test devicescomprising qualitative or quantitative non-lateral flow andnon-microspot array test systems, and (iv) combinations of optical andelectrochemical test devices. For example, the multiple cartridge typesmay include a qualitative lateral flow test device for hCG, aquantitative non-lateral flow test device (e.g., an i-STATelectrochemical cartridge), or a cartridge having both a qualitativelateral flow test and a quantitative non-lateral flow test, e.g., alateral flow hCG combination with an i-STAT CHEM8 cartridge.

In some embodiments, the lateral flow tests performed include hCG, drugsof abuse, and the like. Exemplary non-lateral flow tests include hCG, K,Na, Cl, Ca, Mg, pH, pO2, pCO2, glucose, urea, creatinine, lactate, CKMB,TnI, TnT, BNP, NTproBNP, proBNP, TSH, D-dimer, PSA, PTH, NGAL,galectin-3, AST, ALT, albumin, phosphate, ALP, and the like. Themultiple cartridge types are configured to perform the above-mentionedmultitude of test systems using various biological samples includingurine, whole blood, plasma and serum, both diluted and undiluted, orwith various additives.

In some embodiments, the receiving port 310 may include at least onelocating means 330 for properly positioning cartridges in the housing330 with respect to one or more detectors, e.g., (i) an electricalconnector for connecting to a quantitative electrochemical sensor on thecartridge, and/or (ii) an optical imager for imaging an optical assay inthe cartridge. In some embodiments, the receiving port 310 may beconfigured to sequentially receive the multiple cartridge types.

FIG. 3B shows a lip 340 surrounding the receiving port 310. The lip 340is configured to perform as a light baffle, which substantially blocksexternal light from entering the housing 320 of the reader 300. In someembodiments, the lip 340 may be made of plastic and/or comprise asealant such as rubber. In further embodiments, at least a portion ofthe inside of the reader 300, e.g., an internal coating of the housing320 around the lip 340, may be formed of a material that blocks light orsubstantially absorbs rather than reflects light. Preferably, thematerial is black. In alterative embodiments, the housing 320 may beformed completely opaque by using either black or metal-filled plasticwith a colored outer coating or a black or metalized coating on theinside surface of the housing 320. Further, the housing 320 may beformed to inhibit or prevent light entry at edges between separableparts of the housing 320, e.g., an opaque sealant may be used at theedges. In embodiments where the reader 300 also comprises a lightedgraphic or character display, the display may be isolated from detectionoptics to prevent stray light from the display interfering with thedetection optics. Preferably, the use of instrument self-diagnostic LEDsor similar elements inside the housing 320 is avoided.

FIG. 3C shows a schematic of the lateral flow device 350, e.g., acartridge, where a sample 355 is applied with a capillary or pipette360. In some embodiments, the device 350 may comprise a light baffle 365for engaging with the lip 340 on the reader 300 to inhibit or preventexternal light from entering the receiving port 310. The lateral flowdevice 350 may also comprise a sample wick 370 for receiving the sample355, a conjugate pad 372 for labeling a particular analyte with aconjugate, an optional barcode 375, e.g., a two-dimensional barcode, forproviding information concerning the type of cartridge and/or thepatient, a test window 377 for enabling the reading of the test results,and a waste pad 379 for collecting excess sample and conjugate. In someembodiments, the cartridge may be molded from black or externallymetalized plastic, such that stray light cannot be piped through thecartridge body. Furthermore, a sample closure element 380 may besimilarly treated to prevent stray light passing along the sampleconduit within the cartridge.

FIG. 3D shows the lateral flow device 350 engaged with the reader 300.The reader 300 may comprise one or more detectors for performing theabove described one or more test systems. In some embodiments, as shownin FIG. 3D, the reader 300 may comprise an optical imager 390 comprisinga sensor array, optical elements, and one or more illumination devices.The optical elements may comprise lens 391 and the sensor array maycomprise a camera chip 392, e.g., a charge-coupled device (CCD).

In some exemplary embodiments, the camera chip 392 may, for example,comprise a Canon LiDE210 chip with a 48 bit color resolutionspecification, where the image is digitized to 16 bits (dynamic range of0 to 65,536) in each of the red, green and blue channels of a line scanchip. Aptina array sensors, e.g., MT9V034 (6 μm pixel size) or MT9P031(2.2 μm pixel size), may also be employed. Preferably, the spacialresolution of the CCD is from 4000 to 5000 dpi, e.g., about 4800 dpi,and is able to resolve about from 2 to 10 microns, e.g., about 5.3microns. Thus, for a line feature size of about 0.5 mm, typical of alateral flow test strip, the line width is resolved with an imagequality of about 90 pixels without magnification.

FIGS. 4 and 5 show examples of high resolution color level scale imagesof several test cartridges treated with known levels of hCG. While thetest strips are not necessarily in the focal image plane of the scanner,the test and control lines are imaged sharply enough to be readablequantitatively from 0 to 20 ng/mL hCG. Thus, the imager has aconsiderable depth of field even though the linear sensor array is closeto the object. The illumination and image sharpness across the entireimage is also uniform.

Specifically, FIG. 4 shows a set of six ß-hCG test strips 395 at a rangeof concentrations 0-500 ng/mL as both a standard electronic image 381and using an imager chip 382 within a cartridge housing where controllines 397 and signal lines 396 are located proximate to an imaging areaof the imager chip. The data provided in FIG. 4 demonstrates that theimager used for acquiring barcode information can also be used to readthe bands of a lateral flow device, i.e., distinguish the position andoptical absorbance of the bands. The information is then processed bythe reader to determine one or more of: (i) whether the assay wasperformed properly, (ii) a qualitative result for the presence orabsence of an analyte within a sample (e.g., providing a positive ornegative test result), (iii) a semi-quantitative result for an analyteconcentration with the sample (e.g., wherein the darkness of an opticalsignal correlates to approximate analyte concentration), or (iv) aquantitative result for an analyte concentration within the sample, asdescribed in further detail herein. The device preferably records anddisplays the result. FIG. 5 is similar to FIG. 4 but shows the imagertraces in a single plot of intensity versus pixel position.

In an alternative embodiment, a CCD line camera (e.g., MightexTCN-1304-U) may be used as the camera chip 392. The line camerapreferably comprises a high-performance B/W board-level line camera,based on a single-line 3648-pixel CCD chip with USB 2.0 (480 Mb/s)interface. This type of CCD line camera has several advantages overarea-array counterparts, including high optical linear resolution thatallows capture of two-dimensional (2-D) images by moving the object orthe CCD perpendicularly to the scan line. See, for example, Fan et al;Integrated barcode chips for rapid, multiplexed analysis of proteins inmicroliter quantities of blood, in NATURE BIOTECHNOLOGY, 26, 1373-8,2008, the entirety of which is incorporated herein by reference.

In another alternative embodiment, the camera chip 392 may comprise alinear photodiode or CCD array. In this embodiment, a two-dimensionalimage of the lateral flow test strip or other planar solid phasemultiplex assay test device is acquired by scanning the linear array ina direction perpendicular to its axis. The scanning mechanism preferablyhas micron or nanometer mechanical resolution and carries the lineararray and light sources, which produce a line of light of highuniformity along the length of the linear array, to illuminate the assaytest area immediately in front of the array. The line of light isfocused to give a high brightness, and high spacial resolution andpermit a wide dynamic range of absorbance/reflectance to be detected bythe array. Optionally, the line of light may be pulsed or offset adistance ahead of the array so that the imager can exploit time-resolvedfluorescence assay labels. For example, a time delay of 200 microsecondscan be a achieved by a scanning stage stepping at 100 mm/sec if the lineis offset 20 microns (3 to 4 pixel widths) ahead of the linear array. Alinear microlens array matching pixels 1:1 and overlying the imagingarray can focus on the test area and exclude scattered light from thesource and, in addition, can have an integral interference filter toreject the source wavelengths and pass the emission wavelengths.

In prompt fluorescence and reflectance modes the line is preferablyprojected on the field of view of the microlens-imaging array instead ofahead of the array. Time-resolved detection may also be implemented bypulsing a UV (340-405 nm) light line in the field of view of the lineardetector but holding its exposure gate (shutter) off until 200microsconds after the light pulse is off, followed by integrating for upto 1 millisecond before physically advancing to the next imagingposition. In this case, source light falling on the linear CCDs ispreferably attenuated by incorporating an optical filter in front of theCCD array. Rejection of 6 ODs in the UV range by a long pass filter mayalso be desirable.

In another alternative embodiment, the camera chip 392 may comprise timedelay and integration (TDI) type line-scan array that offersamplification of low light signals and may be used to enhancefluorescence detection sensitivity (e.g., Mightex (Toronto) TCN-1304-U,which in a 1:1 proximity focused design can scan an area over 1 inch(2.54 cm) and is light weight and amenable to inclusion on a portable orhandheld instrument). In another alternative embodiment, the camera chip392 may comprise linear fiber optic arrays that may be used for bothlight source and imaging elements of the line-scan imager.

As also shown in FIG. 3D, the illumination devices may be comprised oflight-emitting diodes (LEDs) 394. For example, multiple wavelength LEDs,e.g., from 405-850 nm, may be used to cover a variety of tests or asingle wavelength LED may be used to increase illumination power.Typical wavelengths for measurements (deltas of absorbance) of variousanalytes are known and depend on the actual assay design, for example467 and 550 nm for total bilirubin, 600 and 550 nm for albumin, 550 and850 nm for total protein, and 400 and 460 nm to distinguish conjugatedand unconjugated bilirubin. Selection of wavelengths such as these maybe achieved by one of ordinary skill in the art.

In alternative embodiments, the illumination may be projected from alaser diode through a cylindrical lens or a fiber optic bundle can beassembled into a linear array. For example, single mode fibers may beused with core diameters of 5 to 10 μm and match reasonably with thepixel size of linear CCD arrays.

An exemplary arrangement of the illumination device and the imagercomprises broadband or/and monochromatic illuminators and a color (RBG)optical imager as spectrophotometer. An advantage of this arrangement isthe ability to detect multiple wavelengths at once. The arrangement alsooffers flexibility in the choice of adding new wavelengths of interest.However, this arrangement has a lower sensitivity due to the RGB maskand the simplicity of the spectrophotometry measurement.

Another arrangement comprises a set of discrete LEDs as illuminators anda gray scale optical imager sensitive to all wavelengths of interest.This arrangement is a simpler design implementation and is moresensitive at a given wavelength; however, the choice of wavelengths isfixed. Specifically, the illumination is provided by a set ofboard-mounted LEDs capable of generating monochromatic excitation insequence (for multi-color assays) to allow detection of the bindingreaction kinetics over time. With respect to the homogeneity ofexcitation, locating a color reference area or areas on the assaysubstrate adjacent to the capture zone eliminates or greatly reduces theneed for homogeneous excitation. In this embodiment, the reference anddetection areas are close enough, e.g., within a few millimeters, suchthat calibration may not be necessary. However, bright and dark imagesmay be accrued prior to testing in order to assess light non-uniformityand fixed pattern corrections to enhance the signal to noise of the testsequence.

In accordance with some aspects of the invention, the camera chip 392 isused to interrogate the barcode 375 and test window 377 of the lateralflow device 350 sequentially or simultaneously. For example, a cartridgefeaturing a qualitative ß-hCG assay based on immuno-chromatographicinstrumented optical detection may be inserted into the reader. Opticaldetection is achieved by using the internal camera acting both as atwo-dimensional barcode reader and as the assay reader. The internalcamera may be integrated into an electromechanical measurement module ofthe reader, which also has a capacity to heat and thermostat thecartridge or portions thereof. Although mainly described in the contextof a ß-hCG assay, the concepts described above are applicable to otherimmuno-chromatographic assays.

FIG. 6 shows an example of an arrangement for the optical imager 390. Inthis embodiment, the LEDs 394 comprise white LEDs 394 a for illuminatingthe barcode 375 on the cartridge and colored LEDs 394 b for illuminatingthe assay area of the strips within the cartridge (e.g., through testwindow 377). A filter 398, e.g., a short pass filter that reflects400-900 nm wavelengths, may be positioned between the LEDs and thecartridge such that the filter 398 reflects the light from the whiteLEDs onto the bar code area and the light from the colored LEDs onto theassay area. An additional LED 399 may also be configured to illuminatethe assay area. For example, the additional LED 399 may be a 350 nm LEDthat illuminates the assay area through the filter 398. The camera chip392 and the lens 395 may be positioned such that a reflected image ofthe barcode area and the assay array is capable of being generated at asame time using the filter 398. This arrangement is capable of providingcompact optics within a relatively small reader device that alsocomprises other functional elements that need to interact with thecartridge, e.g., pump actuators, electrical connectors and the like.

Image Processing to Correct for Illumination Non-Uniformity and toProvide a Qualitative, Semi-Quantitative, or Quantitative Analysis

In preferred embodiments, several phases characterize lateral flow assaytesting and the subsequent processing of information obtained by theimager during and/or after the later flow assay testing. FIG. 7 shows aconceptual representation of the lateral flow assay test strip 130 (asdiscussed with respect to FIG. 1B) in accordance with the preferredembodiments of the present invention. Upon application of a sample tothe test strip 130, the sample flows in the indicated direction bycapillary action and comes into contact with an analyte-specificantibody conjugate zone 135 that may be printed in a soluble form. Acapture antibody zone 140 located downstream of the analyte-specificantibody conjugate zone 135 may be comprised of analyte-specificantibodies immobilized to the chromatographic medium (e.g., paper,nitrocellulose, etc.). Upon the sample now labeled with conjugate fromthe antibody conjugate zone 135, reaching the capture antibody zone 140,any analyte present in the sample may be immobilized by virtue of thecapture antibodies resulting in localization/concentration of thelabeled antigen at the capture antibody zone 140. Material downstreamfrom the capture antibody zone 140 provides a capillary reservoir withwhich to pull sample through the test strip 130.

The presence of analyte in the sample is detected by the presence of theconjugate label in the capture antibody zone 140. Common labels used inthe antibody conjugate zone 135 may include gold colloids (red) and, forexample, blue latex particles, etc. However, there is no requirementthat the label be detectable in the visible range provided that asuitable detector or imager is employed.

As a function of time, several phases of interest taking place on thetest strip 130 can be distinguished. Specifically, the test strip 130 isdry prior to any sample being applied. Wet-up is an initial time periodduring which the sample flows across the length of the strip. During thewet-up phase, the test strip 130 may experience a visible change as aliquid front moves across the chromatographic medium (e.g., paper,nitrocellulose, etc.). Development is the time when labeling and capturetakes place. During the development phase, characteristic bandscorresponding to the capture antibody zone 140 and a control zone 145may become detectable at fixed locations on the test strip 130.

FIGS. 8A-C show examples of images acquired during these various phases(e.g., no sample, wet-up, and development). In FIG. 8A, no sample hasbeen applied. The test strip 130 does not show any visible features(note the absence of characteristic bands). For example, at this stage,the test strip 130 reveals uniform variations associated withillumination and optical data acquisition. Provided the illumination andthe optical characteristics of the measurement module do not changeduring the test phases, these uniformity variations should remainconstant. In FIG. 8B, the liquid front has moved across the length ofthe test strip 130 (e.g., completion of the wet-up phase), anddevelopment has just begun (e.g., initiation of the development phase).Capture and control zones, in the form of characteristic bands acrossthe test strip become detectable. In FIG. 8C, development has takenplace (e.g., completion of the development phase). For example, thecapture and control zones are fully detectable.

FIG. 9 illustrates an exemplary method performed in accordance withpreferred aspects of the present invention for processing a digitalimage of a lateral flow assay strip to generate a qualitative,semi-quantitative, and/or quantitative digital signal. As depicted inFIG. 9, a rectangular subset 150 of the image is defined to include mostof the test strip visible area. The rectangular subset 150 covers mostof the test strip area, but may not include the edges of the test strip130. In lateral flow assays, it maybe typical for the fluid flowcharacteristics on the edge of the test strip to be different from themain area. Not including the edges in the rectangular subset 150 ensuresthat the flow differences are not corrupting the measurements. Pixelintensity may be integrated across the rectangular subset 150 andplotted on a 2-dimensional chart 155. On the 2-dimensional chart 155,the “x” axis represents the distance along the length of the test strip130 in the direction of the flow. The “y” axis represents the integratedintensity for a given distance along the test strip 130. The2-dimensional chart 155 representation of the pixel intensity hasbenefits for accurately determining the presence or absence of theanalyte within the sample (e.g., a qualitative determination), and/ordetermining a concentration of the analyte within the sample (e.g., asemi-quantitative or quantitative determination), as discussed infurther detail below. The integration of the signal is done across thewidth of the test strip 130 in alignment with the capture antibody zone140. For each position along the test strip 130, the resultingsignal-to-noise ratio of the measurement is improved. For strips wherethe capture antibody zones 140 are not visible, the plot indicates tothe illumination uniformity.

FIGS. 10A-C show examples of graphical plots of the assay signalintensity at the three phases of interest (e.g., no sample, wet-up, anddevelopment) for a typical experiment. Specifically, FIG. 10A shows theintegrated signal level along the test strip before the sample isapplied. The shape of the plot is characteristic of the illuminationuniformity along the test strip. FIG. 10B shows the signal level atwet-up. Two dips in the signal are visible at approximately 100 pixelsand 220 pixels. Referring to FIG. 8B, the dips correspond to the captureantibody zones 140 (assay and control bands) where development has justbegun (e.g., completion of the wet-up phase and initiation of thedevelopment phase). FIG. 10C shows the signal level after thedevelopment phase. The two dips in signal intensity have become morepronounced. This is consistent with the intensity of the two bandsvisible in FIG. 8C.

An analysis of the graphical plot depicted in FIG. 10C reveals that aquantitative measurement of the relative intensity of the dips may bedifficult. Without being bound by theory, a main contributing factor tothis difficulty may be the lack of uniform illumination. Accordingly,one of the possible methods to eliminate, or at least reduce, the effectof non-uniform illumination is to subtract a reference image from theimage taken after the development phase. FIGS. 11A-C depict images andgraphs that illustrate results from the subtraction of the image (shownin FIG. 11B) taken after the development phase from the image taken atthe wet-up phase (shown in FIG. 11A). The corresponding 2-dimensionalgraphical plot (shown in FIG. 11C) shows that the effect of lack ofbackground uniformity has been virtually eliminated. Note that a biasmay be introduced to normalize the difference image and ensure that allthe intensity values are positive. Although subtraction and biasadjustment may be performed manually, these steps may be automated viasoftware implementation in accordance with some aspects of the presentinvention. The graphical plot in FIG. 11C features two peaks thatcorrespond to the capture bands. Unlike the plot in FIG. 10C, a ratiomeasurement can easily be made on the processed plot of FIG. 11C toachieve a qualitative, semi-quantitative, and/or quantitativedetermination, as discussed in further detail below.

In alternative or additional embodiments, various choices for areference image to facilitate the qualitative, semi-quantitative, and/orquantitative measurement of the relative amplitude of the signals may beutilized. For example, with respect to FIGS. 11A-11C, a referencesubtraction method based on subtraction of the image taken after thedevelopment phase from the image taken at wet-up phase was described.The use of the image taken after the wet-up phase as a reference imageproduced adequate results for the material and optical characteristicsof the particular lateral flow assay strip. However, for lateral flowassay strips with different characteristics, it may be advantageous touse a different reference image. For example, a subtraction of the imagetaken before the sample is applied from the image taken after thedevelopment phase may be performed. FIGS. 12A-C depict images thatpertain to the subtraction of the image taken after the developmentphase from the image taken before the sample is applied. For thisparticular case, the corresponding 2-dimensional graphical plot in FIG.12C shows that the effect of lack of background uniformity has not beencompletely eliminated. However, the graphical plot in FIG. 12C featurestwo clearly distinguishable peaks that correspond to the capture bandsand a ratio measurement can easily be made to achieve a qualitative,semi-quantitative, and/or quantitative determination, as discussed infurther detail below.

In preferred embodiments, the presence or absence and/or theconcentration of the analyte present in the sample may be determinedand/or quantitated using the graphical plots of signal intensity, asdescribed above. Specifically, one way to quantify the lateral flowassay response is to measure the relative size of the peakscorresponding to the capture antibody and control zones. A ratio of thepeak amplitudes can be made for various concentrations of analyte andfurther processed into response curves. FIG. 13 depicts a plot of theratios between the sample and the control lines for the known analyteconcentration values between 0 and 1000 mIU/mL, as described withrespect to FIGS. 12A-C. The ratio plot shows that the opticalmeasurement set up and the signal processing method described herein iscapable of measuring the concentration of the analyte for the knownconcentrations. Furthermore, the processing method is capable ofmeasuring a response for concentrations below the stated detectionthreshold of the lateral flow assay strips.

Lateral Flow Device

FIG. 14 shows a lateral flow test device 400 in accordance with oneaspect of the invention. In this embodiment, the basic features of acommercial i-STAT cartridge are retained (See, e.g., U.S. PatentPublication No. 2011/0150705, which discloses a non-lateral flow i-STATcartridge) while integrating lateral flow features into the device 400.As shown, the test device 400 comprises lateral flow test systemswithout an electrochemical test system, although in other embodimentsthe test device 400 may incorporate the use of both test systems in thesame device.

As shown, the device 400 comprises an entry port 405 configured toreceive a sample. A sample holding chamber 410 is provided in fluidcommunication with the entry port 405 and is configured to act as aconduit for receiving the sample, optionally via capillary action. Acapillary sample distribution port 412 is provided as an extension ofthe sample holding chamber 410 and is optionally formed into a slot inthe base of the device and may be closed by an optical front cover 415.The optical covering 415 is formed of a transparent material, e.g., a UVtransparent material, and forms the cover of the device 400. Thecapillary sample distribution port 412 also optionally connects to aninlet side of sample channel 420, which may be included, for example, todeliver sample to one or more electrochemical sensors (not shown) on thedevice. The capillary sample distribution port 412 is also in fluidcommunication with a plurality of lateral flow test strips 425positioned within a capillary distribution channel. The device 400 may,for example, comprise “n” number of lateral flow assay strips 425comprising “x” number of assays.

The strips 425 are configured to allow the sample to flow by capillaryaction away from an application site on the test strip. In exemplaryembodiments, as the sample progresses further away from the applicationsite in each respective strip, the sample preferably comes into contactwith a conjugate pad 426 comprising a conjugate label, e.g., ananalyte-specific antibody that is printed in soluble form onto the wickdownstream of the application site. The conjugate label may bind to theanalyte contained within the sample (if present), and forms a sample andconjugate complex. As the sample and conjugate complex progress furtheralong the wick, the complex preferably comes into contact with a capturezone 428, e.g., a chromatographic medium (paper, nitrocellulose etc.)zone located downstream of the conjugate pad. The capture zone may becomprised of analyte-specific antibodies that are immobilized to thewick. Upon reaching the capture zone, any analyte present in the sampleand conjugate complex, will be immobilized by virtue of the captureantibodies resulting in localization/concentration of the labeledantigen at the capture zone. The presence of the analyte is detected bythe presence of the conjugate label in the capture zone. The labels mayinclude, for example, gold colloids or colored latex particles. However,there is no requirement that the label be detectable in the visiblerange provided that a suitable detector/imager is provided, e.g., afluorescent or phosphorescent label activated by a light source alsointegrated into the reader housing may be used. The strips may alsocomprise control zones, which indicate passage of the fluid to thecapture zone 428 ensuring a proper test has been achieved. With asuccessful test, the control zone should indicate a positive resultregardless of whether the sample contains the analyte of interest.

Additional wick material located downstream from the capture zoneprovides a waste pad 430 a, which is configured to pull the sampleacross the wick within the cartridge. In some embodiments, a reservoir430 b is formed as a slot at a terminal end of the strips 425 and isclosed by the optical cover 415. The reservoir 430 b is configured todraw the sample through the strips 425 from the capillary sampledistribution port 412.

As discussed above, the device 400 may also comprise a barcode 435,e.g., a 2D-barcode. The barcode 435 is preferably positioned on thedevice 400 such that a camera chip in the reader device is capable ofimaging the assays on the strips 425 and the barcode 435 sequentially orsimultaneously. For example, the strips 425 and barcode 435 may bepositioned within an imaging area 440 that covers both the strips 425and the barcode 435. The transparency of the optical cover 415 enablesthe imaging area 440 to be illuminated by an illumination device, e.g.,a fiberoptic ring epi-illuminator, and for an image to be taken of theassays on the strips 425 and the barcode 435.

FIG. 14 also shows that the device 400 may comprise at least one mirrorthat forms a 2 pass optical cuvette 445 for detection of total protein(UV spectrum), bilirubin, and hemoglobin (visible spectrum) by use of amicroscale-fiberoptic-coupled UV-VIS diode array spectrometer. A shortporous filter 450 passes the sample, e.g., plasma, into the opticalcuvette 445 from the capillary sample distribution channel. As is shown,conduit 412 may be bifurcated, optionally adjacent optional filter 450,thereby segmenting a blood sample into an optical assay channel and anelectrochemical assay channel.

In preferred embodiments, a detector may be used to determine thepresence or absence of a positive result in the lateral flow assay. Thedetector may be an imager or barcode reader element, e.g., a diode orlaser scanners that function by reflectance, a CCD or CMOS reader orsimilar camera devices, as discussed above in detail. For example, theimager may be integrated into a reader that mates with the lateral flowdevice. When the lateral flow device is inserted into the reader, anillumination source and the imager are activated. As discussed above,the illumination sources can be monochromatic or cover a broad spectrumwithin or outside of the visible range. For example, monochromaticsources used in combination with a color separating barcode sensorenable fluorescence assay detection.

This embodiment also enables the determination of multiple analytes,e.g., drugs of abuse, assayed simultaneously, with each drug having adistinct capture zone. For example, the pattern of “bars” or “dots”detected by the reader establishes which analytes are present and whichare absent. In addition, an imaging area of the device can be dividedinto two distinct zones fulfilling different functions. For example, theimaging area can formed to be about 12×6 mm. One part of the imagingarea may be used to print the barcode information that could be used foridentification of the cartridge type and any additional parametersnecessary to evaluate the result. Another part of the imaging area maybe used to print the arrayed capture zones. In addition, “comparator”zones comprising positive and negative controls may also be printed onthe device in the imaging area. In order to detect contrast betweenpositive and negative controls, or the presence or absence of conjugatelabels by the barcode reader, an automatic gain control feature may beutilized to optimize a dynamic range of the acquired image to maximize anumber of levels. A threshold value determination may be used tocharacterize each capture site as “positive” or “negative.”

The lateral flow device may be assembled comprising several “wicking”elements including a sample deposition element that filters, forexample, blood cells from the sample so that the assay proceeds withplasma. The conjugate zone may also be applies as a separate elementpreviously impregnated with conjugate or other sample treatmentreagents. Furthermore, the device may be assembled as a single opticalassay cartridge or integrated with electrochemically based assays in thesame cartridge, as discussed in further detail below.

FIGS. 15A-15C show side, plan, and front views, respectively, of afolded lateral flow cartridge 460 for a single lateral flow strip. Thecartridge 460 is shown engaged with reader 470. FIGS. 16A-16C showperspective and side views of a bottom portion 480 and a top portion 485of a lateral flow cartridge in accordance with another embodiment of theinvention. In some embodiments, the top portion 485 is configured toreceive application of a sample through a sample entry port 470 of thecartridge. The sample entry port 470 is in fluid communication with anapplication site on the sample wick or strip 490, as described abovewith respect to FIG. 14.

Microspot Array Device

As an alternative to reading a lateral flow device, the imager withinthe reader may be configured to read a microspot array within acartridge. In this embodiment, individual reagents are immobilized asspots in an array on a substantially planar surface within thecartridge, as discussed above. Each spot in the array is assigned aspecific coordinate (row x; line y) and has preselected dimensions,e.g., circular with radii in the range 10-1000 μm. This information maybe either pre-programmed into the reader or can be decoded from acorresponding barcode, which may be read before, after or simultaneouslywith the reading of the microspot array. Consequently, the image capturesoftware can identify each spot and determine from the spot intensity,for example, one or more of the presence or absence of the analyte,analyte concentration, or a calibration signal. Adjacent areas of thetest device that are accessible to the imager can also provide a flatfield correction grid which acts as an integrated internal assay set ofreference spots.

FIGS. 17A and 17B show test devices 500 (cartridges) having microspotarrays in accordance with some embodiments of the invention. FIG. 17Ashows a cartridge having a microspot array without electrochemicalfeatures. FIG. 17B shows that the basic electrochemical features of acommercial i-STAT cartridge may be retained (See, e.g., U.S. Pat. Nos.5,096,669; 5,447,440; 6,750,053; 7,419,821; and 7,682,833, and U.S.Publication No. 2011/0150705, the entireties of which are incorporatedherein by reference and which disclose non-lateral flow i-STATcartridges) while integrating microspot array features into the device500. The individual reagents may be printed and immobilized as spotsusing known methods. See, e.g., Cozzette et al., U.S. Pat. No.5,200,051, the entirety of which is incorporated herein by reference.Thus, the test device 500 may comprise microspot array test systemswithout electrochemical test systems (FIG. 17A) or the test device 500may incorporate the use of both test systems on the same device (FIG.17B).

As shown in FIG. 17A, the device 500 comprises an entry port 505configured to receive a sample. A sample holding chamber 510 is providedin fluid communication with the entry port 505 and is configured to actas a conduit for the sample. A capillary sample distribution port 512 isprovided as an extension of the sample holding chamber 510 and is formedinto a slot in the base closed by an optical cover 515. The opticalcovering 515 is formed of a transparent material, e.g., a UV transparentmaterial, and forms the front cover of the device 500. The capillarysample distribution port 512 is in fluid communication with a microspotarray 525 positioned within a microspot array chamber. The microspotarray 525 (e.g., comprising capture antibodies or antigens) comprises aplurality of spots for multiplex assays. For example, the microspotarray 525 may have an approximate capacity for about 600 spots, whichwould permit high level protein and DNA multiplex assays. After thesample has passed through the microspot array, e.g., through wicking,the sample may be delivered through a waste conduit to waste chamber530.

In some embodiments, the device 500 may also comprise a barcode 535,e.g., a 2D-barcode. The barcode 535 is positioned on the device 500 suchthat a camera chip in the reader is capable of imaging of the microspotarray 525 and the barcode 535 at a same time. For example, microspotarray 525 and barcode 535 may be positioned within an imaging area 540that covers both the microspot array 525 and the barcode 535. Thetransparency of the optical cover 515 enables the imaging area 540 to beilluminated by an illumination device, e.g., a fiberoptic ringepi-illuminator, and for an image to be taken of the microspot array 525and the barcode 535.

In embodiments in which the microarray test system and theelectrochemical test system are comprised on the same test device, themicrospot array chamber may be configured in series or in parallel withan electrochemical sensor channel. The test device 500 of FIG. 17B, forexample, is substantially as described above in connection with FIG.17A, but also includes the same type of fluidic designs and capabilitiesrendering its suitable also for electrochemical assays, and showselectrochemical and optical sensing systems operating in series. Thedevice 500 comprises an entry port 505 configured to receive a sample. Asample holding chamber 510 is provided in fluid communication with theentry port 505 and is configured to act as a conduit for the sample.After optionally being pushed through a capillary stop 514 the sample isdelivered to an electrochemical sensing conduit 516 in which animmunoassay is formed on one or more electrodes 513. Afterelectrochemical sensing, the resulting fluid is directed through conduit517 to microspot array 525 positioned within a microspot array chambersuch the sample is directed to the microspot array. The microspot array525 (e.g., comprising capture antibodies or antigens) may have aplurality of spots for multiplex assays. Thus, in this embodiment, thesample may be first processed in the electrochemical section and thenpushed into the optical section. In this embodiment, the solution maycomprise both electrochemical and optical substrates for imaging. As theelectrochemical detection is completed, the substrate is delivered tothe microspot array chamber subsequent to the electrochemical detection.The sample remains in the microspot array chamber during imageinterrogation in chemiluminescence and precipitating fluorescencesubstrate based assays. After the sample has passed through themicrospot array, the sample may be delivered through a waste conduit 518to waste chamber 530.

In another embodiment, sample fluid from the holding chamber 510 isdivided into two streams. One stream is directed to the electrochemicalsensor via conduit 520 and another stream is directed to the microspotarray in a manner similar to FIG. 17A. The resulting streams undergoseparate analysis by electrochemical and optical processes, as describedabove, and may be separately directed to waste chamber, or may becombined and directed to the waste chamber together. FIG. 25, discussedbelow, shows a similar embodiment allowing parallel optical andelectrochemical detection.

FIG. 18 shows a fluorescent image of a test device with a microspotarray. In one aspect, the monochromatic image illustrated in FIG. 18 iswhat is viewed by the imager. The calibration standards contained withinthe array may also be used as grid landmarks for auto-alignment of theimager. In some embodiments, ultra-bright conjugate labels may be usedfor low cost or low sensitivity imagers.

For calibration, it is possible to print a dilution series comprising,for example, IgA, IgG, IgM, IgD, IgE within the microarray that containsantigens or antibodies for the array of test analytes. The printedreagents can generate standard curves for each within the sample.Aspects of the calibration may include homogeneity of the illuminationintensity. The calibration spots may be collocated next to the assayspots to minimize variability. In an alternative embodiment, an imagemay be captured prior to the test cycle for calibration purposes.Advantageously, any factors that influence the array test spots willalso affect the calibration spots. Therefore, common influences such asrheumatoid factor, lipemia, hemolysis, intravenous fluids,immunoglobulins and the like, which can change the slope of thecalibration curves are corrected, thus providing a quantitativemeasurement for each of the tests. Another advantage of this assay isthe inclusion of replicates (e.g., three as shown for each calibratorand test in FIG. 18).

In some embodiments, off axis illumination arrangements as discussedabove with respect to FIG. 6 may reduce or eliminate specularreflections allowing various substrates with different reflectivityproperties to be used in the device. For example, the support substratesmay include paper, micro/nano-porous filters, glass, plastic, silicon,alumina, polymer gels, and the like. These substrates may beincorporated into various kinds of microfluidic structures of thedevice, e.g., the structures shown in FIG. 17B.

Another advantage of the microspot array is the ability to multiplex.For example, different classes of patient responses may be determined bya color of the emitted light. One of ordinary skill in the art wouldunderstand that many analyte targets may be chosen, e.g., classes ofdrugs, different classes of cytokines and inflammatory markers.

The present embodiment may also comprise nanoparticle phosphor(time-resolved fluorescent) immunoassays, e.g., using SeradynEu-chelate-loaded time-resolved assays. Two light sources may be used,e.g., a pulsed blue LED and a xenon lamp. Fluorescein, ruthenium chelateand platinum-porphyrin can be excited in the blue and UV range, whereaseuropium chelate is excited only in the UV. While fluorescein is promptand disappears in a few nanoseconds, Ru emission disappears after 50μsec, Pt after 250 μsec, and Eu chelate reaches its peak emission at 250μsec and lasts several milliseconds. One of ordinary skill in the artwould understand that this is just one example of a three-levelfluorescence multiplex labeling format without the need for multipleoptical filters, and the disclosed invention is not limited by thisarrangement.

FIG. 19 shows a fluorescent image of an exemplary test device with aquantum dot array.

Cartridge with Lateral Flow Fluidics and Conductimetric Fluid PositionDetection

In some embodiments, the cartridges or devices may further comprisedetection components for conductance-based determination of the positionof a fluid within a microfluidic circuit of the cartridge and lateralflow fluidic components for the active movement of the fluid through thecartridge based on the determination of the position of the fluid. Forexample, gold electrodes on a flexible or rigid printed circuit may belocated within the channels of the microfluidic circuit at pointsimportant to the functional control of the fluidic device. A change inconductance/impedance between pairs of electrodes occurs when the fluidis in contact with the pair and forms a contiguous fluid bridge betweenthem. Thus a measured signal consistent with a fluid partially orentirely between the pair of electrodes means that its position is knownby the analytical system (e.g., the reader and/or the cartridge).

The analytical system may comprise a pump whose pressure or displacementis under logic or computer control (e.g., computing device 215,discussed below). The pump may be connected to the microfluidic circuitand may be used to automatically move the fluid until aconductance/impedance change indicates the fluid is bridging a chosenpair of electrodes, and thus occupies a known position. In embodiments,the pump may be a pneumatic pump, a hydraulic pump, a syringe, or thelike.

FIGS. 20 and 21 provide two examples (e.g., lateral flow and microspotarray, respectively) for implementing conductance-based determinationand active lateral flow movement in a cartridge test system. Inaccordance with some embodiments of the invention, the cartridge 600 maycomprise a pump 605, a fluid-containing reservoir 610, a microfluidiccircuit 615 with electrodes 620 at position control points, a lateralflow (chromatographic) test strip or a flow-through microspot assaychamber 630, and an imager, e.g., a camera chip or photosensor, forrecording the assay using optical measurement.

In alternative embodiments, the position and/or total volume of thefluid can be determined by using the imager or an imaging sensor. Forexample, the sample fluid channels can be formed to route the fluid tothe imaging area (as discussed above with respect to FIGS. 14 and 17A/B)for subsequent imaging by the imager and the determination ofpositioning within the microfluidic circuit. Specifically, the fluid maybe imaged as it passes through the imaging area, which allows a precisedetermination of the timing of the fluid motion. One of ordinary skillin the art would understand that other means for detection may beutilized in the above-disclosed systems, e.g., amperometric-baseddetermination may also be used for detecting the position of the fluid.

Cartridge with Combined Pneumatic and Lateral Flow Fluidic Features

In some embodiments, the invention relates to cartridges, as describedabove, that further comprise pneumatic fluidic actuation to provide afurther degree of control over the various steps of the assays, e.g.,the processes described with respect to the lateral flow device in FIG.14 or 17A/B. As discussed above, the lateral flow test strips generallyoperate in a passive mode (i.e., the sample flows through a supportmatrix of the wick via capillary action). Due to the nature of passivesystems, however, there is limited or no control over the time domain asthe fluid or sample passes through the wick. Consequently there islimited or no control over the amount of mixing that may occur betweenthe sample and the conjugate or bound antibodies in the capture zone.Therefore, in some embodiments, a pump, as described above, may be usedwith the cartridges to actively control fluid migration through animmuno-chromatographic assay. For example, the cartridge may comprise adisplacement device, e.g., an air bladder, that is actuated by a pump tomix the sample with the conjugate and antibodies of the capture zone orcontrol the time domain.

FIG. 22 shows a design of an assay cartridge with a displacement device,e.g., an air bladder, and an immuno-chromatographic assay. One ofordinary skill in the art would understand that FIG. 22 does notrepresent a specific assay geometry, rather it represents a layout ofelements that may be utilized in the cartridge for active fluid control,e.g., a test device with pneumatic mixing of a sample with a labeledantibody and a lateral flow capture zone. In some embodiments, the totalsample volume may be between approximately 5 μL and 500 μL, thus thedepicted sample chamber would accommodate such volumes in practicalembodiments, and the adjoining conduits would be suitably sized.

As shown in FIG. 22, the cartridge comprises an inlet port 640 that isused to collect the sample. The sample may be provided, for example, inthe form of urine, serum, plasma, or whole blood. The cartridge furthercomprises a sample fill chamber 645, a labeling zone 650, achromatographic assay capture zone 655, a waste chamber 660, an airbladder 675, and fluidic channels 680 that provide a fluid connectionbetween the various components of the cartridge. First, the cartridge isinserted into a reader, e.g., through the universal cartridge receivingport. The chromatographic assay capture zone is then imaged by an imagerchip, e.g., a 2D barcode reader, and the air bladder 675 is actuated bya pump, e.g., a pneumatic pump, a hydraulic pump, a syringe, or thelike. In some embodiments, the barcode reader and the pump may becontrolled by embedded software within the reader, e.g., the computingdevice 215.

In operation, once the cartridge is inserted through receiving port, thesample accumulates in the sample fill chamber 645. A restriction,optionally a capillary stop, located at the end of the fill chamber 645may provide an indication when the fluid level is sufficient. After thesample fluid is deposited in the cartridge, a latch (not shown) may beused to close the port. Once the latch is closed, the cartridge isinserted in the reader and an automated measurement cycle begins. Themeasurement cycle may be comprised of several phases. First, the airbladder 675 is activated to push the sample fluid to a labeling zone 650where the sample comes into contact with an analyte-specific antibodyconjugate, which preferably has been printed in a soluble form onto thewalls of labeling zone 650. This dissolves the antibody into the sampleand allows for binding of the analyte-specific antibody conjugate withthe analyte. The air bladder 675 is then actuated to sequentially pushand pull the fluid sample through the fluidic channels 680 on thecartridge. The resulting oscillatory motion facilitates mixing of thesample fluid with the antibody conjugate. Once the sample fluid has beensatisfactorily mixed with the antibody conjugate, the air bladder 675 isactuated to push the fluid to the chromatographic assay capture zone 655where the analyte-specific antibodies are immobilized to thechromatographic medium (e.g., a porous plug made of paper,nitrocellulose, etc.). The timing of these steps may be controlled bythe software described with respect to FIG. 2. In some embodiments,completion of all the steps may take approximately two to twenty minutesdepending on the assay and sample type.

Upon reaching the capture zone 655, any analyte present in the sample,now labeled with conjugate, will be immobilized by virtue of the captureantibodies resulting in localization of the labeled antigen at thecapture zone. Optionally, the air bladder 675 can be actuated tosequentially push and pull the sample fluid across the capture zone 655to ensure optimal capture of the antibodies. Once the analyte has beensatisfactorily captured, the air bladder 675 can be optionally actuatedto push the sample fluid into the waste chamber 660, leaving behind thecaptured antibodies. In some embodiments, a wash fluid contained withina wash fluid chamber 685 may be pushed through the chromatographic assaycapture zone 655 to flush away components of the sample fluid that arenot necessary for result generation or can be a source of interference,e.g., red blood cells. The wash fluid is preferably located in arupturable pouch within the device. See, e.g., Lauks et al., U.S. Pat.No. 5,096,669, the entirety of which is incorporated herein byreference.

For detection of the labeled antigen, an illumination source and animager located in the reader are activated, and one or more images areacquired of the chromatographic assay capture zone 655 and optionally anadjacent area comprising a barcode. The image or series of images areanalyzed by the reader, e.g., the computing device 215. An automatedsoftware analysis derives a positive or negative result based on thecharacteristics of the acquired image or series of images. In someembodiments, the barcode may contain assay information, e.g., testidentification, calibration data, color references, test cycle controlparameter, expiration data and the like. Once the image acquisition iscompleted, the cartridge may be released from the instrument anddisposed.

This embodiment has significant advantages over passive lateral flowimmuno-chromatographic devices. For example, it enables instrument datacapture of the test results, which are then available for transmissionto a Laboratory Information System (LIS) or a Hospital InformationSystem (HIS) for recordkeeping and billing.

The present embodiment also advantageously minimizes the time it takesto perform the test and reduces the opportunity for a user-inducederror. For tests that are performed manually on typical lateral flowdevices, a guideline for wait time after application of the sample onthe wicking element before reading the assay is provided by themanufacturer. The wait time includes the time for capillary flow of thesample along the wicking element, the sample labeling time and theconjugate label capture time onto the area where the measurement isperformed. For most tests currently commercially available, a wait timebetween several seconds and several minutes is typical. By using activefluid control, the present embodiment reduces the transit times fromsample collection to the labeling zone and from the labeling zone to thecapture zone. In addition to reducing the labeling and capture timeswhen compared to passive capillary flow methods, active fluid mixingadvantageously improves sensitivity.

This embodiment further allows customizing the measurement cycle for thefluid sample type. For example a ß-hCG assay is compatible with bothwhole blood and urine samples. Labeling and capture phases of themeasurement cycle can be optimized depending on the sample type by meansof the active fluidic control. Such optimization is not typicallypossible for traditional lateral flow assays where assay completiontimes typically vary depending on the sample type and are based oncompromises regarding the porosity and other properties of the lateralflow matrix. Further, imaging of the capture zone may optionally beperformed at various times during the test cycle, resulting in a seriesof time resolved images. Analysis of the images may be conducted by theanalysis software to derive the rate of color change in the captureregion. From the knowledge of the rate of the change from controlledexperiments during manufacture, the reader may be programmed with ananticipated completion time window for data collection for each sampletype, blood, plasma, serum, urine etc. This adaptive data acquisitioncan lead to shorter measurement cycles.

FIG. 23 shows another embodiment of a test device with pneumatic mixingof a labeled antibody with a sample further comprising multiple lateralflow capture zones 687. The present embodiment may also comprisemultiplex assays. FIG. 24 shows an alternative embodiment of a testdevice with pneumatic mixing of a labeled antibody with a sample furthercomprising lateral flow capture zones 689 in multiple channels 690.Active fluidic control is extended to multiple labeling andchromatographic assay capture zones for a multiplexed assay of the samefluid sample. FIG. 24 further shows a manifold 700 that separates thesample fluid into two separate labeling zones 705. Another manifold 710collects the sample fluid after detection in the capture chamber andallows disposal of the sample fluid in the waste chamber 712. Thisdesign can be extended to any number of channels that can practicallyfit on a cartridge and any number of capture zones that can be imaged bythe imager, e.g., a barcode reader, within the reader.

The immuno-chromatographic assay cartridge may also incorporate multiplelabeling and chromatographic assay capture zones in parallel. In thisembodiment, the cartridge comprises a single labeling chamber wheremultiple analyte-specific antibody conjugates are printed in adissolvable form. When the fluid sample comes in contact with theantibody conjugates they dissolve and the analytes of interest becomelabeled with their specific conjugate. The labeled analytes are thenpushed to the chromatographic assay capture chamber by the pneumaticpump assembly. The capture chamber features multiple chromatographicassay capture zones sequentially arranged in the direction of the flow.Each analyte, now labeled with the conjugate, is immobilized to aspecific area of the chromatographic medium.

In another embodiment, the cartridge layouts described above may includeadditional fluidic channels for delivering the fluid sample to othertypes of sensors. The other sensors are not limited to electrochemicalsensors, but can include fiber optic sensors, waveguide sensors, surfaceacoustic wave sensors, surface plasmon wave sensors, thermal sensors andthe like, for measuring designated sample properties. FIG. 25 shows thisembodiment of the test device with pneumatic mixing of a labeledantibody with a sample further comprising lateral flow capture zones 720in one channel and electrochemical detectors 725 in another channel. Inthis embodiment, the fluidic path is divided into two distinct paths.One path delivers the sample to the labeling and capture zones. Theother path delivers the sample to an electrochemical sensors area. Inthis embodiment, the reader comprises an electrical connector, asdiscussed above with respect to FIGS. 3A-3D, for engaging theelectrochemical sensors with the control circuitry within the reader.

Test Sample Separator

In some embodiments, cells or particles within a test sample mayinterfere with the flow of a sample through the wicking matrix of alateral flow device. Therefore, the above-described cartridges maycomprise a sample separator to separate the test sample, e.g., blood maybe separated into cells and plasma. FIG. 26 shows a cartridge or devicecomprising a sample separator. The sample separator may comprise aseparation chamber that utilizes gravity to assist in separation of thetest sample. One of ordinary skill in the art would understand that FIG.26 does not represent a specific geometry; rather it represents aconcept of the elements found in the cartridge.

As shown in FIG. 26, the cartridge may comprise an inlet port 800 thatis used to collect a test sample. In the case of the ß-hCG assay, urine,plasma/serum, or whole blood can be used. The cartridge furthercomprises a sample fill chamber 810 that serves as a labeling zone, asedimentation chamber 820, a lateral flow assay strip 825 with achromatographic assay capture zone 830, a waste chamber 840, an airbladder 850, and fluidics channels 860 to connect the components of thecartridge. Once inserted in the reader, the chromatographic assaycapture zone 830 can be imaged by an internal imager and the air bladder850 can be actuated by a pump. Both the imager and the pump may becontrolled by embedded software in the reader.

Once the sample is inserted through the inlet port 800, the biologicalsample accumulates in the sample fill chamber 810 and comes in contactwith labeling conjugates already printed in the chamber 810. Theconjugates dissolve in the sample fluid and the analyte of interestbecomes labeled. Subsequently, the air bladder 850 may be activated topush the labeled sample fluid to a sedimentation chamber 820 where thesample, e.g., blood, is allowed to rest and sediment. Sedimentation cannaturally happen by gravity or can be accelerated by a chemical process(e.g., addition of a clumping agent). The geometry of the chamber can beoptimized to retain clumped blood cells while allowing the serum tocontinue flowing. During the period of time used for sedimentation, theorientation and motion of the reader is monitored by an internalaccelerometer to ensure no unwanted motion perturbs the sedimentationprocess. Inertial forces due to unwanted motion or excessive angleorientation can prevent sedimentation at the bottom of the chamber.

Acceleration and angle deviation from gravity can be measured by ameasuring device, e.g., Analog Devices ADXL345. The ADXL345 is a small,thin, ultra-low power, 3-axis accelerometer with high resolution(13-bit) measurement at up to ±16 g. The measuring device measures thestatic acceleration of gravity in tilt-sensing applications, as well asdynamic acceleration resulting from motion or shock. The ADXL345 highresolution (3.9 mg/LSB) enables measurement of inclination changes lessthan 1.0°. Threshold values can be assigned for both dynamicacceleration and angle deviation from rest position during thesedimentation phase of the test. If the accelerometer detects values fordynamic acceleration or angle that exceed threshold values, a computerin the reader, e.g., computing device 215 (discussed below), can take aseries of actions that can include displaying a warning message to theoperator, modifying the test cycle accordingly (e.g., additional resttime), correcting the measurement value or issuing an error code andcanceling the test altogether.

The air bladder 850 is further activated to push the labeled andseparated sample fluid to a sample application zone on the lateral flowassay strip 825. Once the labeled and separated sample fluid comes intocontact with the application zone on the lateral flow assay strip,capillary forces pull the fluid toward the chromatographic assay capturezone 830 where the analyte-specific antibodies are immobilized to thechromatographic medium. Upon reaching the capture zone 830, any analytepresent in the sample, now labeled with conjugate, will be immobilizedby virtue of the capture antibodies resulting inlocalization/concentration of the labeled antigen at the capture zone830.

Illumination sources and an imager (e.g., located in the reader) arecontrolled to acquire one or several images of the chromatographic assaycapture zone 830. The image or the series of images are analyzed by thesoftware of the reader. An automated software analysis derives apositive or negative result based on the characteristics of the acquiredimage or series of images. Once the image acquisition is completed, thecartridge may be released and disposed.

Although the embodiment of a sample separator has been described withinthe context of a lateral flow assay to allow sedimentation of the bloodcells in a dedicated part of the fluidic channels, the concept ofmonitoring the angle and dynamic motion of the reader during test withan internal sensor (e.g., multi-axis accelerometer such as the ADXL345or other) is applicable to any assay (e.g., electro-chemical or optical)that can benefit from stability requirements. Generally, if a stabilityrequirement exists as part of a testing cycle on a cartridge, a dynamicmotion or angle sensor can be activated to measure motion and positionparameters during the critical phases of the measurement cycle. Ifunacceptable motion is detected, an internal logic can modify thetesting cycle accordingly or initiate a warning communication to theuser or both.

System Environment

In view of the foregoing, it will be appreciated by those of ordinaryskill in the art that in some aspects the present invention is embodiedin a single device or apparatus (e.g., a reader device or a testcartridge), a system, a method or a computer program product.Accordingly, in some embodiments, the present invention relates tocertain hardware, software (including firmware, resident software,micro-code, etc.) or embodiments combining software and hardware, whichmay be referred to herein as a “circuit,” “module” or “system.”Furthermore, in some embodiment, the present invention may take the formof a computer program product embodied in one or more computer readablemedium(s) having computer readable program code embodied thereon. Forexample, such software, systems and computer readable medium(s) may beincorporated into the reader device or test cartridges of the invention.

Any combination of one or more computer readable medium(s) may beutilized. The computer readable medium may be a computer readable signalmedium or a computer readable storage medium. A computer readablestorage medium may be, for example, but is not limited to, anelectronic, magnetic, optical, electromagnetic, infrared, orsemiconductor system, apparatus, or device, or any suitable combinationof the foregoing. A non-limiting list of specific examples of thecomputer readable storage medium includes: an electrical connectionhaving one or more wires, a portable computer diskette, a hard disk, arandom access memory (RAM), a read-only memory (ROM), an erasableprogrammable read-only memory (EPROM or Flash memory), an optical fiber,a portable compact disc read-only memory (CD-ROM), an optical storagedevice, a magnetic storage device, or any suitable combination of theforegoing. In the context of this document, a computer readable storagemedium may be any tangible medium that can contain, or store a programfor use by or in connection with an instruction execution system,apparatus, or device, e.g., a reader device or a cartridge.

A computer readable signal medium may include, for example, a propagateddata signal with computer readable program code embodied therein, forexample, in baseband or as part of a carrier wave. Such a propagatedsignal may take any of a variety of forms, including, but not limitedto, electro-magnetic, optical, or any suitable combination thereof. Acomputer usable storage memory can be any physical storage device suchas random access memory (RAM) or a read-only memory (ROM) to name a fewexamples.

Program code embodied on a computer readable medium may be transmittedusing any appropriate medium, including but not limited to wireless,wireline, optical fiber cable, RF, etc., or any suitable combination ofthe foregoing.

Computer program instructions may also be stored in the computerreadable medium that can direct a computer, other programmable dataprocessing apparatus, or other devices to function in a particularmanner, such that the instructions stored in the computer readablemedium produce an article of manufacture including instructions thatimplement the function/act specified in the flowchart and/or blockdiagram block or blocks.

The computer program instructions may also be loaded onto a computer,other programmable data processing apparatus, or other devices to causea series of operational steps to be performed on the computer, otherprogrammable apparatus or other devices to produce a computerimplemented process such that the instructions which execute on thecomputer or other programmable apparatus provide processes forimplementing the functions/acts specified in the flowchart and/or blockdiagram block or blocks.

FIG. 2 shows an illustrative environment 200 for managing the processesin accordance with some embodiments of the invention. To this extent,the environment 200 includes a server or other computing system 210 thatcan perform the processes described herein. In particular, the computingsystem 210 includes a computing device 215 such as a cartridge readerdevice and a cartridge 217 (e.g., a cartridge comprising optical and/orelectrochemical assay systems). An example of such a system is theaforementioned i-STAT system sold by Abbott Point of Care Inc. Thei-STAT portable blood analysis system may comprise a Wi-Fi-enabledreader device that works in conjunction with single-use blood testingcartridges that contain sensors for various analytes. The computingdevice 215 can be resident on a network infrastructure or computingdevice of a third party service provider (any of which is generallyrepresented in FIG. 2).

The computing device 215 also includes a processor 220, memory 220A, anI/O interface 230, and a bus 240. The memory 220A can include localmemory employed during actual execution of program code, bulk storage,and cache memories which provide temporary storage of at least someprogram code in order to reduce the number of times code should beretrieved from bulk storage during execution. In addition, the computingdevice includes RAM, ROM, and an operating system (O/S).

The computing device 215 may be in communication with an external I/Odevice/resource 250 and an external storage system 220B. For example,the I/O device 250 can comprise any device that enables an individual tointeract with the computing device 215 (e.g., user interface) or anydevice that enables the computing device 215 to communicate with one ormore other computing devices using any type of communications link. Theexternal I/O device/resource 250 may be for example, a handheld device,PDA, handset, keyboard, etc.

In general, the processor 220 executes computer program code (e.g.,program control 260), which can be stored in the memory 220A and/orstorage system 220B. Moreover, in accordance with some aspects of theinvention, the program control 260 controls at least one control and/ormeasurement module 270 to perform the processes described herein. Thecontrol module and/or measurement 270 can be implemented as one or moreprogram code in the program control 260 stored in memory 220A asseparate or combined modules. Additionally, the control and/ormeasurement module 270 may be implemented as separate dedicatedprocessors or a single or several processors to provide the function ofthis tool. While executing the computer program code, the processor 220can read and/or write data to/from memory 220A, storage system 220B,and/or I/O interface 250. The program code executes the processes of theinvention. The bus 240 provides a communications link between each ofthe components in the computing device 215.

In some embodiments, the control and/or measurement module 270 mayperform optical and/or electrochemical tests in conjunction with thecartridge 217 comprising optical and/or electrochemical assay systems.For example, in accordance with some aspects of the invention, thecontrol and/or measurement module 270 can operate sensors of the opticaland/or electrochemical assay systems within the cartridge 217 to providequalitative, semi-quantitative, and/or quantitative measurements of ananalyte within a test sample, and display such measurements to a user.In another embodiment, upon insertion of a cartridge 217 device into thereader device, the control and/or measurement module 270 may operate oneor more feature of the reader device to determine whether the cartridge217 is an optical cartridge, an electrochemical cartridge, or both.

EXAMPLE

The present invention may be better understood in view of the followingnon-limiting example.

A series of experiments were conducted to determine the ability of theoptical detection method to quantify analyte concentrations. Biologicalsamples consisting of male urine spiked with β-hCG at variousconcentrations were used on lateral flow assay strips designed toindicate a response when the analyte concentration exceeds 25 mIU/mL.Images were collected during the experiments and processed to produce adifference image and signal. FIGS. 27A-D depict images of the developedassay strips at 0, 15, 100 and 1000 mIU/mL of β-hCG. The bar graphlocated below each image indicates the concentration of analyte comparedto the qualitative detection threshold of the lateral flow assays. Thetriangle indicates the level for which the lateral flow assay strips aredesigned to indicate a positive result. Wet-up images were obtained foreach experiment and used to create a difference image and signal. FIGS.28A-D depict the processed images that were obtained by subtracting theimages taken after development from the images taken during wet-up.FIGS. 29A-D depict corresponding graphical plots when the pixel valuesare integrated across the highlighted rectangular area.

FIG. 29A shows the 2-D graphical plot for the case where there is noβ-hCG in the sample. Only one peak is visible on the plot. The visiblepeak corresponds to the control band. FIG. 29B shows the graphical plotof pixel intensity for the case where the sample had a knownconcentration of 15 mIU/mL. Two peaks are visible on the plot. Thelarger peak corresponds to the control band. The smaller one correspondsto the capture zone of the analyte. FIG. 29C shows the case where thesample had a known concentration of 100 mIU/mL. Two peaks are visible onthe plot. The larger peak corresponds to the control band. The smallerpeak corresponds to the capture zone of the analyte. Note that therelative size of the two peaks is different when the analyteconcentrations are different. Specifically, the peak corresponding tothe analyte capture zone gets larger as the concentration increases.FIG. 29D corresponds to the case where the analyte concentration is 1000mIU/mL. Two peaks are still visible as was the case with the analyteconcentrations shown in FIGS. 29B and 29C. However, in FIG. 29D wherethe analyte concentration is 1000 mIU/mL, the peak corresponding to thecapture zone has become larger than the peak corresponding to thecontrol band. Specifically, it is typical for the intensity of thecapture zones to become larger than the control zones when the analyteconcentration becomes very high.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. Accordingly, it isintended that the scope of the present invention be limited solely bythe scope of the following claims.

We claim:
 1. An analyte test cartridge comprising: a sample entry portto receive a test sample; a sample holding chamber connected to thesample entry port; a conduit fluidically connected to the sample holdingchamber, the conduit comprising a labeling zone comprising a labeledantibody specific for an analyte and comprising a label, and a capturezone located downstream from the labeling zone and comprising a porousplug with a capture antibody for the analyte; and a displacement devicecapable of mixing the test sample with the labeled antibody and to movethe test sample through at least a portion of the analyte test cartridgefrom the labeling zone to the capture zone.
 2. The analyte testcartridge of claim 1, wherein the porous plug is a lateral flow strip ora microspot array.
 3. The analyte test cartridge of claim 1, wherein thelabel is an optically detectable moiety.
 4. The analyte test cartridgeof claim 1, wherein the porous plug is positioned to align with anoptical detector in an instrument that receives and engages the analytetest cartridge.
 5. The analyte test cartridge of claim 1, furthercomprising a plasma separator positioned in at least a portion of thesample holding chamber proximate to the sample entry port.
 6. Theanalyte test cartridge of claim 5, wherein the test sample is wholeblood when received at the sample entry port and the test sample isplasma subsequent to the sample holding chamber.
 7. The analyte testcartridge of claim 1, wherein: the labeled antibody is part of a beadreagent; and the displacement device is capable of moving the testsample into the conduit, mixing the bead reagent with the test sample toform a complex of the analyte with the bead reagent, and moving thecomplex to the capture antibody to capture the complex.
 8. The analytetest cartridge of claim 1, wherein the labeled antibody is capable ofdissolving into the test sample.
 9. The analyte test cartridge of claim1, further comprising a second conduit connected to the sample holdingchamber and comprising an electrochemical sensor.
 10. The analyte testcartridge of claim 1, further comprising a cover comprising atransparent material positioned for optical detection of a complex,captured by the capture antibody, of the analyte with the labeledantibody.
 11. An analyte test cartridge comprising: a sample entry portto receive a test sample; a sample holding chamber connected to thesample entry port; a conduit fluidically connected to the sample holdingchamber, the conduit comprising a labelling zone comprising a pluralityof labeled antibodies specific for a plurality of analytes, and one ormore capture zones, the one or more capture zones comprising a pluralityof capture antibodies for the plurality of analytes; and a displacementdevice capable of mixing the test sample with the plurality of thelabeled antibodies and to move the test sample through at least aportion of the analyte test cartridge from the labeling zone to the oneor more capture zones.
 12. The analyte test cartridge of claim 11,wherein the one or more capture zones comprise multiple porous plugs,with each of the plurality of capture antibodies located on a differentporous plug.
 13. The analyte test cartridge of claim 12, wherein themultiple porous plugs are multiple lateral flow strips.
 14. The analytetest cartridge of claim 11, wherein the one or more capture zonescomprise a porous plug comprising multiple areas with each of theplurality of capture antibodies.
 15. The analyte test cartridge of claim14, wherein the porous plug is a lateral flow test strip.
 16. Theanalyte test cartridge of claim 14, wherein the one or more capturezones comprise a microspot array.
 17. The analyte test cartridge ofclaim 11, wherein at least some of the plurality of labeled antibodiesare optically labeled.
 18. The analyte test cartridge of claim 11,wherein the one or more capture zones are positioned to align with anoptical detector in an instrument that receives and engages the analytetest cartridge.
 19. The analyte test cartridge of claim 11, furthercomprising a plasma separator positioned in at least a portion of thesample holding chamber proximate to the sample entry port.
 20. Theanalyte test cartridge of claim 19, wherein the test sample is wholeblood when received at the sample entry port and the test sample isplasma subsequent to the sample holding chamber.